Purified compounds are required for testing and analysis protocols applied in many scientific fields. Purification of a compound involves separating a desired component or components from a mixture that contains additional components or impurities. Chromatography methods can be applied to fractionate a mixture into separate components. In liquid chromatography, a sample containing a number of components to be separated is injected into a system flow and directed through a chromatographic column. The column separates the mixture by differential retention into its separate components. The components elute from the column as distinct bands separated in time.
A typical high performance liquid chromatography (HPLC) system includes a pump for delivering a fluid (the “mobile phase”) at a controlled flow rate and composition, an injector to introduce a sample solution into the flowing mobile phase, a chromatographic column that contains a packing material or sorbent (the “stationary phase”), and a detector to detect the presence and amount of the sample components in the mobile phase leaving the column. When the mobile phase passes through the stationary phase, each component of the sample typically emerges from the column at a different time because different components in the sample typically have different affinities for the packing material. The presence of a particular component in the mobile phase exiting the column can be detected by measuring changes in a physical or chemical property of the eluent. By plotting the detector signal as a function of time, response “peaks” corresponding to the presence and quantities of the components of the sample can be observed.
Preparative HPLC is a convenient way to isolate and purify a quantity of a compound for further studies or use. Depending on the specific application, preparative separations can be performed using large columns and sample sizes, or may be performed using small columns for smaller volume collection of components. A common distinction between preparative and analytical HPLC is that for preparative HPLC, the sample components are collected after purification, whereas for analytical HPLC, the sample components are simply detected and quantified.
HPLC systems sometimes require that a sample be diluted before the sample is injected into the mobile phase flowing to the chromatography column. The solvent used to dilute the sample may interfere with the ability to obtain a desired chromatographic resolution. Generally, it is preferable to keep a sample focused at the head of the chromatographic column; however, strong solvents can limit the retention of the sample and instead promote the release of the sample as it enters the column. In some separations, the result may be two chromatographic peaks and, in other separations, the sample may simply flow with the solvent through the column.